Targeted lipidomics

Targeted lipidomic analysis was performed using a LC ESI-QQQ MS/MS model 6490 (Agilent Technologies, Melbourne, Australia). 652 lipid species were detected, 14 were acylcarnitines of the fatty acyl (FA) category; 46 were cholesterol derivatives of the category of sterol lipids (ST) and 157 were sphingolipids (SP) including ceramides, gangliosides, sphingomyelin and sulfatides. Of the glycerolipids (GL) category 67 lipids were detected, 20 of them diacylglycerols and the rest of them triacylglycerols, mostly unsaturated. Most of the lipids were part of the glycerophospholipid (GP) category; concretely 367 lipid species were detected; 124 with ethanolamine (being 73 ether lipids, 14 of them with an alkyl ether bond and 59 plasmalogens); 193 with choline (being 62 ether lipids, 32 of them plasmalogens as well and 32 plasmanyl species); 11 with serine; 35 with inositol and only 4 lipid species conjugated with another molecule of glycerol. The only lipid detected in the prenol (PR) category was ubiquinone. Sample tissues were randomized prior to lipid extraction. Lipids were extracted in a single phase chloroform:methanol (2:1) procedure as previously described (Meikle et al., 2011). Briefly, to 10 μL of the stock tissue homogenates, 200 μL of chloroform/methanol (2:1, v/v) were added together with 10 μL of internal standards in chloroform/methanol (1:1, v/v). Lipid standards stock solutions were prepared by dissolving lipid standards (Table S1) in chloroform:methanol (1:1,v/v) at 100 pmol except for cholesterol that was 10,000 pmol, cholesteryl ester 1,000 pmol, and sphingomyelin and diacylglycerol that were at 200 pmol. Finally, for the dihydroceramides and hexosylceramides the working solutions were at 50 pmol. The mixture was mixed for 10 min on a rotary mixer, sonicated in a water bath (18–24°C) for 30 min, left to stand on the bench for 20 min and then centrifuged at 16,000 × g at 20°C for 10 min. The supernatant was transferred to a 96-well plate and dried under a stream of nitrogen gas at 40°C. Samples were reconstituted with 50 μL H₂O-saturated 1-butanol and sonicated for 10 min. Then, 50 μL of 10 mM ammonium formate in methanol was added. The extract was centrifuged at 1,700 × g at 20°C for 5 min. The recovery efficiencies of the lipid extraction method for each lipid subclass were previously published in other work (Alshehry et al., 2015). Finally, supernatant was transferred into a 0.2 mL glass insert with Teflon insert cap for analysis by LC ESI-MS/MS. Technical quality controls (internal lipid standard mix solution) were injected every 10 samples as well as lipid extraction quality controls (plasma samples with internal lipid standard mix solution) every 15 samples. One microliter of lipid extract was applied onto ZORBAX eclipse plus C18 column, 2.1 × 100 mm 1.8 μm, (Agilent Technologies) heated to 60°C and the auto-sampler regulated to 25°C. Flow rate was 400 μL/min with solvent A composed of 10 mM ammonium formate in acetonitrile-water-isopropanol (50:30:20, v/v) and solvent B composed of 10 mM ammonium formate in acetonitrile-water-isopropanol (9:1:90, v/v). The gradient started at 10% of mobile phase B, eached 100% B in 11 min and held for 1 min. Finally, the system was switched back to 10% of mobile phase B and was equilibrated for 3 min. Data was collected in the multiple reaction monitoring scan type and the capillary voltage was set at 3,500 V. Positive polarity of electrospray ionization was set using N2 at 20 psi as nebulizer gas (17 L/min, 150°C) and the sheath gas parameters were flow at 10 L/min and temperature at 200°C. For all the standard lipid species the cell accelerator voltage was 5 volts, except for the Sph(d17:1) that was 4 volts and fragmentor was 380 volts. The conditions for tandem mass spectrometry quantification of the 652 lipid species detected by the targeted lipidomic analysis are reported in Table S2. To determine the acyl composition for each glycerophospholipid species, we performed MS2 experiments in pooled plasma samples on our chromatography set-up (Supplementary Figure 1). Phosphatidylethanolamine and phosphatidylinositol species were fragmented in negative ionization mode and product ions corresponding to their acyl chains were examined. Phosphatidylcholine and sphingomyelin species were examined in positive ion mode in the presence of lithium acetate, and the product ions of the [M+Li]+ precursor was used to determine their acyl composition (Hsu and Turk, 2003). Moreover, to determine whether observed peaks were either ether lipids or plasmalogens, we utilized our quality control plasma extracts. Plasma extracts were dried down under nitrogen and exposed to HCl vapor for 5 min. Sample was then reconstituted in the same solvent (butanol:methanol, 1:1). Injection of quality control samples with and without exposure to HCl was used to determine peaks corresponding to plasmalogens, where the susceptibility of the vinyl-ether bond to acid results in complete hydrolysis (Supplementary Figure 2). Technical quality control coefficient variation (CV) was 7.6% across measured lipids suggesting a good technical reproducibility.